Efficacy of Allogeneic and Xenogeneic Exosomes for the Treatment of Canine Atopic Dermatitis: A Pilot Study.

Canine atopic dermatitis (CAD) is a genetically predisposed inflammatory pruritic skin disease. The available treatments for CAD have several adverse effects and vary in efficacy, indicating the need for the development of improved treatments. In this study, we aimed to elucidate the therapeutic effects of allogeneic and xenogeneic exosomes on CAD. Six laboratory beagle dogs with CAD were randomly assigned to three treatment groups: control, canine exosome (cExos), or human exosome (hExos) groups. Dogs in the cExos and hExos groups were intravenously administered 1.5 mL of cExos (5 × 1010) and hExos (7.5 × 1011) solutions, respectively, while those in the control group were administered 1.5 mL of normal saline three times per week for 4 weeks. Skin lesion score and transepidermal water loss decreased in cExos and hExos groups compared with those in the control group. The exosome treatments decreased the serum levels of inflammatory cytokines (interferon-γ, interleukin-2, interleukin-4, interleukin-12, interleukin-13, and interleukin-31) but increased those of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-ß), indicating the immunomodulatory effect of exosomes. Skin microbiome analysis revealed that the exosome treatments alleviated skin bacterial dysbiosis. These results suggest that allogeneic and xenogeneic exosome therapy may alleviate CAD in dogs.

Does gut brain axis has an impact on Parkinson's disease (PD)?

Parkinson's Disease (PD) is becoming a growing global concern by being the second most prevalent disease next to Alzheimer's Disease (AD). Henceforth new exploration is needed in search of new aspects towards the disease mechanism and origin. Evidence from recent studies has clearly stated the role of Gut Microbiota (GM) in the maintenance of the brain and as a root cause of various diseases and disorders including other neurological conditions. In the case of PD, with an unknown etiology, the GM is said to have a larger impact on the disease pathophysiology. Although GM and its metabolites are crucial for maintaining the normal physiology of the host, it is an undeniable fact that there is an influence of GM in the pathophysiology of PD. As such the Enteroendocrine Cells (EECs) in the epithelium of the intestine are one of the significant regulators of the gut-brain axis and act as a communication mediator between the gut and the brain. The communication is established via the molecules of neuroendocrine which are said to have a crucial part in neurological diseases such as AD, PD, and other psychiatry-related disorders. This review is focused on understanding the proper role of GM and EECs in PD. Here, we also focus on some of the metabolites and compounds that can interact with the PD genes causing various dysfunctions in the cell and facilitating the disease conditions using bioinformatical tools. Various mechanisms concerning EECs and PD, their identification, the latest studies, and available current therapies have also been discussed.

Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production. In this study, we evaluated the utilization of thermostable basic fibroblast growth factor (TS-bFGF) with MSC culture and exosome production. MSCs cultured with TS-bFGF displayed superior proliferation, as evidenced by cell cycle analysis, compared with wild-type bFGF (WT-bFGF). Stemness was assessed through mRNA expression level and colony-forming unit (CFU) assays. Furthermore, nanoparticle tracking analysis (NTA) measurements revealed that MSCs cultured with TS-bFGF produced a greater quantity of exosomes, particularly under three-dimensional culture conditions. These produced exosomes demonstrated substantial anti-inflammatory and wound-healing effects, as confirmed by nitric oxide (NO) assays and scratch assays. Taken together, we demonstrate that utilization of TS-bFGF for WJ-MSC-derived exosome production not only increases exosome yield but also enhances the potential for various applications in inflammation regulation and wound healing.

Efficient improvement of the proliferation, differentiation, and anti-arthritic capacity of mesenchymal stem cells by simply culturing on the immobilized FGF2 derived peptide, 44-ERGVVSIKGV-53.

INTRODUCTION: The stem cell microenvironment has been evidenced to robustly affect its biological functions and clinical grade. Natural or synthetic growth factors, especially, are essential for modulating stem cell proliferation, metabolism, and differentiation via the interaction with specific extracellular receptors. Fibroblast growth factor-2 (FGF-2) possesses pleiotropic functions in various tissues and organs. It interacts with the FGF receptor (FGFR) and activates FGFR signaling pathways, which involve numerous biological functions, such as angiogenesis, wound healing, cell proliferation, and differentiation. OBJECTIVES: Here, we aim to explore the molecular functions, mode of action, and therapeutic activity of yet undetermined function, FGF-2-derived peptide, FP2 (44-ERGVVSIKGV-53) in promoting the proliferation, differentiation, and therapeutic application of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) in comparison to other test peptides, canofin1 (FP1), hexafin2 (FP3), and canofin3 (FP4) with known functions. METHODS: The immobilization of test peptides that are fused with mussel adhesive proteins (MAP) on the culture plate was carried out via EDC/NHS chemistry. Cell Proliferation assay, colony-forming unit, western blotting analysis, gene expression analysis, RNA-Seq. analysis, osteogenic, and chondrogenic differentiation capacity were applied to test the activity of the test peptides. We additionally utilized three-dimensional (3D) structural analysis and artificial intelligence (AI)-based AlphaFold2 and CABS-dock programs for receptor interaction prediction of the peptide receptor. We also verified the in vivo therapeutic capacity of FP2-cultured hWJ-MSCs using an osteoarthritis mice model. RESULTS: Culture of hWJ-MSC onto an FP2-immobilized culture plate showed a significant increase in cell proliferation (n = 3; *p < 0.05, **p < 0.01) and the colony-forming unit (n = 3; *p < 0.05, **p < 0.01) compared with the test peptides. FP2 showed a significantly upregulated phosphorylation of FRS2α and FGFR1 and activated the AKT and ERK signaling pathways (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Interestingly, we detected efficient FP2 receptor binding that was predicted using AI-based tools. Treatment with an AKT inhibitor significantly abrogated the FP2-mediated enhancement of cell differentiation (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Intra-articular injection of FP2-cultured MSCs significantly mitigated arthritis symptoms in an osteoarthritis mouse model, as shown through the functional tests (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), modulation of the expression level of the pro-inflammatory and anti-inflammatory genes, and improved osteochondral regeneration as demonstrated by tissue sections. CONCLUSION: Our study identified the FGF-2-derived peptide FP2 as a promising candidate peptide to improve the therapeutic potential of hWJ-MSCs, especially in bone and cartilage regeneration.

Role of Telomeres and Telomerase in Parkinson's Disease-A New Theranostics?

Parkinson's disease (PD) is a complex condition that is significantly influenced by oxidative stress and inflammation. It is also suggested that telomere shortening (TS) is regulated by oxidative stress which leads to various diseases including age-related neurodegenerative diseases like PD. Thus, it is anticipated that PD would result in TS of peripheral blood mononuclear cells (PBMCs). Telomeres protect the ends of eukaryotic chromosomes preserving them against fusion and destruction. The TS is a normal process because DNA polymerase is unable to replicate the linear ends of the DNA due to end replication complications and telomerase activity in various cell types counteracts this process. PD is usually observed in the aged population and progresses over time therefore, disparities among telomere length in PBMCs of PD patients are recorded and it is still a question whether it has any useful role. Here, the likelihood of telomere attrition in PD and its implications concerning microglia activation, ageing, oxidative stress, and the significance of telomerase activators are addressed. Also, the possibility of telomeres and telomerase as a diagnostic and therapeutic biomarker in PD is discussed.

Human Endogenous Retrovirus-H-Derived miR-4454 Inhibits the Expression of DNAJB4 and SASH1 in Non-Muscle-Invasive Bladder Cancer.

Although most human endogenous retroviruses (HERVs) have been silenced and lost their ability to translocate because of accumulated mutations during evolution, they still play important roles in human biology. Several studies have demonstrated that HERVs play pathological roles in numerous human diseases, especially cancer. A few studies have revealed that long non-coding RNAs that are transcribed from HERV sequences affect cancer progression. However, there is no study on microRNAs derived from HERVs related to cancer. In this study, we identified 29 microRNAs (miRNAs) derived from HERV sequences in the human genome. In particular, we discovered that miR-4454, which is HERV-H-derived miRNA, was upregulated in non-muscle-invasive bladder cancer (NMIBC) cells. To figure out the effects of upregulated miR-4454 in NMIBC, genes whose expression was downregulated in NMIBC, as well as tumor suppressor genes, were selected as putative target genes of miR-4454. The dual-luciferase assay was used to determine the negative relationship between miR-4454 and its target genes, DNAJB4 and SASH1, and they were confirmed to be promising target genes of miR-4454. Taken together, this study suggests that the upregulation of miR-4454 derived from HERV-H in NMIBC reduces the expression of the tumor suppressor genes, DNAJB4 and SASH1, to promote NMIBC progression.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.

Improved Wound Healing and Skin Regeneration Ability of 3,2'-Dihydroxyflavone-Treated Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Flavonoids enhance the self-renewal and differentiation potential of mesenchymal stem cells (MSCs) and have therapeutic activities, including regenerative, anti-oxidative, and anti-inflammatory effects. Recent studies have revealed that MSC-derived extracellular vesicles (MSC-EVs) have therapeutic effects on tissue regeneration and inflammation. To facilitate further research on the therapeutic potential of MSC-EVs derived from flavonoid-treated MSCs, we surveyed the production of EVs and their therapeutic applications in wound regeneration. MSCs treated with flavonoids enhanced EV production twofold compared with naïve MSCs. EVs produced by MSCs treated with flavonoids (Fla-EVs) displayed significant anti-inflammatory and wound-healing effects in vitro. The wound-healing capacity of EVs was mediated by the upregulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Interestingly, the protein level of p-ERK under inhibition of MEK signals was maintained in Fla-EV-treated fibroblasts, suggesting that Fla-EVs have a higher therapeutic potential than naïve MSC-EVs (Cont-EVs) in wound healing. Moreover, the in vivo wound closure effect of the Fla-EVs showed significant improvement compared with that of the flavonoid-only treatment group and the Cont-EVs. This study provides a strategy for the efficient production of EVs with superior therapeutic potential using flavonoids.

Alterations and Co-Occurrence of C-MYC, N-MYC, and L-MYC Expression are Related to Clinical Outcomes in Various Cancers.

Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

Ductal delivery of extracellular vesicles promote the recovery from salivary gland inflammation.

Salivary gland dysfunction worsens the quality of life, but treatment for restoration of salivary gland function is limited. Although previous reports have demonstrated the therapeutic potentials of extracellular vesicles (EVs) in different preclinical models, the role of EVs in salivary glands remains elusive. Furthermore, little is known about the roles of salivary gland-derived EVs in tissue repair or regeneration compared to other EVs. In this study, EVs secreted from salivary gland-derived mesenchymal stem cells (sgMSCs) were comparatively analyzed with those from Wharton's jelly-derived MSC (wjMSCs). sgMSCs secreted more significant amounts of EVs than wjMSCs, and salivary gland epithelial cells showed a more efficient uptake of sgMSC-EVs than wjMSC-EVs. The possibility of immune regulation was tested via macrophage polarization and LPS-induced epithelial inflammation, resulting in an M1-to-M2 shift and reversal of acinar-to-ductal metaplasia by sgMSC-EV. Furthermore, the roles of sgMSC-EV-mediated immune regulation and tissue repair were clarified in vivo via retroductal delivery of sgMSC-EVs in a mouse model of obstructive sialadenitis. Collectively, our data demonstrate the superior role of sgMSC-EVs in the recovery from salivary gland inflammation and injury and suggest EVs as therapeutic tools for salivary gland dysfunction.

TMBIM6-mediated miR-181a expression regulates breast cancer cell migration and invasion via the MAPK/ERK signaling pathway.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) has been reported to regulate cell death pathways and is overexpressed in several types of cancers. In this study, we investigated whether high expression of TMBIM6 in breast cancer was significantly associated with cancer invasiveness. Knockdown of TMBIM6 reduced proliferation and migration of invasive breast cancer cells through downregulation of the MAPK/ERK signaling pathway. Moreover, we suggested that expression of miR-181a was significantly suppressed upon TMBIM6 knockdown. In contrast, overexpression of TMBIM6 significantly increased cell invasion and migration through up-regulation of mesenchymal markers and matrix metalloproteinase-9 (MMP-9) and enhanced activation of the MAPK/ERK signaling pathway. We also observed that up-regulation of TMBIM6 significantly increased the expression of miR-181a by TMBIM6-mediated pathway. TMBIM6 and miR-181a-mediated ERK activation induced the expression of Snail-1 and Snail-2 in FOSL-1/C-JUN-dependent manner. Overall, our data demonstrated that TMBIM6-induced miR-181a up-regulation plays an important role in the efficient modulation of migration and invasion of breast cancer cells.

Anti-oncogenic effects of dutasteride, a dual 5-alpha reductase inhibitor and a drug for benign prostate hyperplasia, in bladder cancer.

BACKGROUND: The incidence of bladder cancer (BCa) is approximately four times higher in men than in women. To develop effective BCa treatments, there is an urgent need to understand the differences in the BCa control mechanisms based on gender. Our recent clinical study showed that androgen suppression therapy using 5α-reductase inhibitors and androgen deprivation therapy affects BCa progression, but the underlying mechanisms are still unknown. METHODS: mRNA expression levels of the androgen receptor (AR) and SLC39A9 (membrane AR) in T24 and J82 BCa cells were evaluated by reverse transcription-PCR (RT-PCR). The effect of dutasteride, a 5α-reductase inhibitor, in BCa progression was determined in cells transfected with control and AR-overexpressing plasmids. In addition, cell viability and migration assays, RT-PCR, and western blot analysis were performed to analyze the effect of dutasteride on BCa in the presence of testosterone. Finally, steroidal 5α-reductase 1 (SRD5A1), one of the dutasteride target genes, was silenced in T24 and J82 BCa cells using control and shRNA-containing plasmids, and the oncogenic role of SRD5A1 was evaluated. RESULTS: Dutasteride treatment led to significant inhibition of the testosterone-induced increase dependent on AR and SLC39A9 in cell viability and migration of T24 and J82 BCa cells and induced alterations in the expression level of cancer progression proteins, such as metalloproteases, p21, BCL-2, NF-KB, and WNT in AR-negative BCa. Furthermore, the bioinformatic analysis showed that mRNA expression levels of SRD5A1 were significantly higher in BCa tissues than in normal paired tissues. A positive correlation between SRD5A1 expression and poor patient survival was observed in patients with BCa. Also, Dutasteride treatment reduced cell proliferation and migration via blocking the SRD5A1 in BCa. CONCLUSIONS: Dutasteride inhibited testosterone-induced BCa progression dependent on SLC39A9 in AR-negative BCa and repressed oncogenic signaling pathways, including those of metalloproteases, p21, BCL-2, NF-KB, and WNT. Our results also suggest that SRD5A1 plays a pro-oncogenic role in BCa. This work provides potential therapeutic targets for the treatment of BCa.

The Orphan GPR50 Receptor Regulates the Aggressiveness of Breast Cancer Stem-like Cells via Targeting the NF-kB Signaling Pathway.

The expression of GPR50 in CSLC and several breast cancer cell lines was assessed by RT-PCR and online platform (UALCAN, GEPIA, and R2 gene analysis). The role of GPR50 in driving CSLC, sphere formation, cell proliferation, and migration was performed using shGPR50 gene knockdown, and the role of GPR50-regulated signaling pathways was examined by Western blotting and Luciferase Assay. Herein, we confirmed that the expression of G protein-coupled receptor 50 (GPR50) in cancer stem-like cells (CSLC) is higher than that in other cancer cells. We examined that the knockdown of GPR50 in CSLC led to decreased cancer properties, such as sphere formation, cell proliferation, migration, and stemness. GPR50 silencing downregulates NF-kB signaling, which is involved in sphere formation and aggressiveness of CSLC. In addition, we demonstrated that GPR50 also regulates ADAM-17 activity by activating NOTCH signaling pathways through the AKT/SP1 axis in CSLC. Overall, we demonstrated a novel GPR50-mediated regulation of the NF-κB-Notch signaling pathway, which can provide insights into CSLC progression and prognosis, and NF-κB-NOTCH-based CSLC treatment strategies.

Bioactive peptides for boosting stem cell culture platform: Methods and applications.

Peptides, short protein fragments, can emulate the functions of their full-length native counterparts. Peptides are considered potent recombinant protein alternatives due to their specificity, high stability, low production cost, and ability to be easily tailored and immobilized. Stem cell proliferation and differentiation processes are orchestrated by an intricate interaction between numerous growth factors and proteins and their target receptors and ligands. Various growth factors, functional proteins, and cellular matrix-derived peptides efficiently enhance stem cell adhesion, proliferation, and directed differentiation. For that, peptides can be immobilized on a culture plate or conjugated to scaffolds, such as hydrogels or synthetic matrices. In this review, we assess the applications of a variety of peptides in stem cell adhesion, culture, organoid assembly, proliferation, and differentiation, describing the shortcomings of recombinant proteins and their full-length counterparts. Furthermore, we discuss the challenges of peptide applications in stem cell culture and materials design, as well as provide a brief outlook on future directions to advance peptide applications in boosting stem cell quality and scalability for clinical applications in tissue regeneration.

Advanced 3D dynamic culture system with transforming growth factor-ß3 enhances production of potent extracellular vesicles with modified protein cargoes via upregulation of TGF-ß signaling.

INTRODUCTION: Mesenchymal stromal cells (MSCs) release extracellular vesicles (MSC-EVs) containing various cargoes. Although MSC-EVs show significant therapeutic effects, the low production of EVs in MSCs hinders MSC-EV-mediated therapeutic development. OBJECTIVES: Here, we developed an advanced three-dimensional (a3D) dynamic culture technique with exogenous transforming growth factor beta-3 (TGF-ß3) treatment (T-a3D) to produce potent MSC-EVs. METHODS: Our system enabled preparation of a highly concentrated EV-containing medium for efficient EV isolation and purification with higher yield and efficacy. RESULTS: MSC spheroids in T-a3D system (T-a3D spheroids) showed high expression of CD9 and TGF-ß3, which was dependent on TGF-ß signaling. Treatment with EVs produced under T-a3D conditions (T-a3D-EVs) led to significantly improved migration of dermal fibroblasts and wound closure in an excisional wound model. The relative total efficacy (relative yield of single-batch EVs (10-11-fold) × relative regeneration effect of EVs (2-3-fold)) of T-a3D-EVs was approximately up to 33-fold higher than that of 2D-EVs. Importantly the quantitative proteomic analyses of the T-a3D spheroids and T-a3D-EVs supported the improved EV production as well as the therapeutic potency of T-a3D-EVs. CONCLUSION: TGF-ß signalling differentially regulated by fluid shear stress produced in our system and exogenous TGF-ß3 addition was confirmed to play an important role in the enhanced production of EVs with modified protein cargoes. We suggest that the T-a3D system leads to the efficient production of MSC-EVs with high potential in therapies and clinical development.

Rapid production method with increased yield of high-purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide.

Extracellular vesicles (EVs) are nano-sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large-scale production of high-purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30-fold) in EV production with markedly high purity (approximately 45-fold). These EVs (TS-eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA-AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS-eEV production induced by eMTDΔ4, indicating that the eMTDΔ4-mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS-eEVs. The present study may lead to considerable advances in EV-based drug development and production of stem cell-derived EVs for cell therapy.

TTYH3 Modulates Bladder Cancer Proliferation and Metastasis via FGFR1/H-Ras/A-Raf/MEK/ERK Pathway.

Tweety family member 3 (TTYH3) is a calcium-activated chloride channel with a non-pore-forming structure that controls cell volume and signal transduction. We investigated the role of TTYH3 as a cancer-promoting factor in bladder cancer. The mRNA expression of TTYH3 in bladder cancer patients was investigated using various bioinformatics databases. The results demonstrated that the increasingly greater expression of TTYH3 increasingly worsened the prognosis of patients with bladder cancer. TTYH3 knockdown bladder cancer cell lines were constructed by their various cancer properties measured. TTYH3 knockdown significantly reduced cell proliferation and sphere formation. Cell migration and invasion were also significantly reduced in knockdown bladder cancer cells, compared to normal bladder cancer cells. The knockdown of TTYH3 led to the downregulation of H-Ras/A-Raf/MEK/ERK signaling by inhibiting fibroblast growth factor receptor 1 (FGFR1) phosphorylation. This signaling pathway also attenuated the expression of c-Jun and c-Fos. The findings implicate TTYH3 as a potential factor regulating the properties of bladder cancer and as a therapeutic target.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4'-Dihydroxyflavone, U0126, and N-Acetyl Cysteine.

Background and Objectives: Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Methods and Results: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4'-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4'-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4'-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4'-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs. Conclusions: Our results showed that co-treatment of 3, 4'-DHF, U0126, and/or NAC cumulatively regulated osteogenesis in eADSCs, suggesting that 3, 4'-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Superior therapeutic activity of TGF-ß-induced extracellular vesicles against interstitial cystitis.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disease characterized by incapacitating pelvic pain. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered key mediators of the paracrine action of MSCs and show better biological activities than the parent MSCs, especially in the bladder tissue, which may be unfavorable for MSC survival. Here, we produced MSC-EVs using advanced three-dimensional (a3D) culture with exogenous transforming growth factor-ß3 (TGF-ß3) (T-a3D-EVs). Treatment with T-a3D-EVs led to significantly enhanced wound healing and anti-inflammatory capacities. Moreover, submucosal layer injection of T-a3D-EVs in chronic IC/BPS animal model resulted in restoration of bladder function, superior anti-inflammatory activity, and recovery of damaged urothelium compared to MSCs. Interestingly, we detected increased TGF-ß1 level in T-a3D-EVs, which might be involved in the anti-inflammatory activity of these EVs. Taken together, we demonstrate the excellent immune-modulatory and regenerative abilities of T-a3D-EVs as observed by recovery from urothelial denudation and dysfunction, which could be a promising therapeutic strategy for IC/BPS.

New therapeutic approach with extracellular vesicles from stem cells for interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating chronic disorder characterized by suprapubic pain and urinary symptoms such as urgency, nocturia, and frequency. The prevalence of IC/BPS is increasing as diagnostic criteria become more comprehensive. Conventional pharmacotherapy against IC/BPS has shown suboptimal effects, and consequently, patients with end-stage IC/BPS are subjected to surgery. The novel treatment strategies should have two main functions, anti-inflammatory action and the regeneration of glycosaminoglycan and urothelium layers. Stem cell therapy has been shown to have dual functions. Mesenchymal stem cells (MSCs) are a promising therapeutic option for IC/BPS, but they come with several shortcomings, such as immune activation and tumorigenicity. MSC-derived extracellular vesicles (MSC-EVs) hold numerous therapeutic cargos and are thus a viable cell-free therapeutic option. In this review, we provide a brief overview of IC/BPS pathophysiology and limitations of the MSC-based therapies. Then we provide a detailed explanation and discussion of therapeutic applications of EVs in IC/BPS as well as the possible mechanisms. We believe our review will give an insight into the strengths and drawbacks of EV-mediated IC/BPS therapy and will provide a basis for further development. [BMB Reports 2022; 55(5): 205-212].